The abundant heme-binding protein of the liver, probably identical with Z-protein or liver fatty acid-binding protein, has an apparent molecular weight of 14,000 Da and is presumably involved in the intracellular transport of a variety of compounds. The cellular and subcellular distribution of HBP in the liver was studied in adult male and female rats by postembedding immunocytochemistry using the protein A-gold technique. By light microscopic examination heme-binding protein is present exclusively in parenchymal cells and not found in the sinusoidal lining cells or other cells in portal tracts. Immunoreactivity for heme-binding protein is uniformly strong throughout the liver lobule in female rats but is markedly reduced in the pericentral region in male animals. By immunoelectron microscopy heme-binding protein immunoreactivity is localized in cytoplasm and nuclear matrix. The mitochondria and peroxisomes and the secretory apparatus are free of the label. In nuclei, gold labeling is confined to the interchromatin region (euchromatin) and nucleoli; condensed chromatin (heterochromatin) and nucleolus-associated chromatin are negative. The subcellular localization was substantiated by radioimmunoassay and immunoblotting of nuclear and cytosolic fractions. Immunoblotting shows that the heme-binding protein-like immunoreactive protein in the nucleus has a slightly larger molecular weight than that in the cytoplasm.